Afterward, anti\B220 staining in PBS + 1% BSA for 30 min or actin cytoskeleton staining with phalloidin diluted in PBS + 5% FBS + 0.3% Triton X\100 was performed for 1 h at RT. at 0C with a fluorescent anti\goat Ig antibody. This allowed us to distinguish between B cells having membrane\attached beads (positive for the anti\goat Ig antibody) and B cells that experienced completely internalized beads (unfavorable for anti\goat Ig staining). Using this approach, we could clearly determine by confocal microscopy that follicular B cells were able to phagocytose particles of 1 1 and 3 m in diameter, presenting the typical rearrangement of the plasma membrane round the particles while remaining unfavorable for Dimebon 2HCl the anti\goat Ig staining (Fig ?(Fig1A).1A). In order to quantify this phagocytic process, we applied the same theory using circulation cytometry. Using this method, we could monitor the percentage of B cells with phagocytosed beads according to their unfavorable staining for the anti\goat Ig antibody, as well as the different number of phagocytosed beads, up to 5, according to the stepwise increase in fluorescent intensity in the bead Dimebon 2HCl fluorescence Dimebon 2HCl channel (Fig ?(Fig1B).1B). This method allowed us to calculate a phagocytic index that displays the percentage of B cells that have phagocytosed beads and the number of phagocytosed beads per cell (Fig ?(Fig1B).1B). Using this method, we could corroborate that follicular B cells can phagocytose 1 and 3 m beads by a BCR\specific process actively, since it is usually blocked at 0C (Fig EV1A). Furthermore, we showed that B cells incubated at 37C and permeabilized with detergent became all positive for anti\goat Ig staining, indicating that anti\goat Ig unfavorable B cells experienced truly phagocytosed the beads (Fig EV1B). The phagocytic ability of follicular B cells experienced a size limitation since they were basically unable to internalize 10 m particles (Fig ?(Fig1C).1C). Furthermore, beads internalization by B cells was inhibited by cytochalasin D and latrunculin A, two inhibitors of the rearrangement of the actin cytoskeleton, and by PP2, an inhibitor of tyrosine kinases of the src family (Fig EV1C), thus suggesting that it is a bona fide phagocytic process triggered by BCR signaling. These data show that, contrary to general belief 11, 12, 33, na?ve B cells are able to phagocytose antigen\coated particles in a BCR\driven process. Open in a separate window Physique 1 Follicular B cells phagocytose particulates antigens through a RhoG\dependent mechanism Confocal section of follicular B cells in the process of phagocytosing 1 and 3 m beads coated with anti\IgM. Purified follicular B cells were incubated with 1 or 3 m fluorescent beads coated with a goat anti\mouse anti\IgM for 1 h at 37C and afterward stained with an anti\goat 488 antibody on ice to distinguish cells with attached or already internalized beads. Beads are shown in green, SLC2A3 the extracellular Dimebon 2HCl staining with anti\goat IgG in reddish, and the cortical actin cytoskeleton in blue. Completely phagocytosed beads, unfavorable for anti\goat IgG, are indicated with an arrow, and non\phagocytosed beads are indicated with an asterisk. Circulation cytometry plots of WT\ and RhoG\deficient B cells incubated for 1 h with 1 m fluorescent beads coated with anti\IgM antibody and stained afterward extracellularly with anti\goat 488, as in (A). The phagocytic index was calculated according to the stepwise increase in the beads mean fluorescence intensity and lack of anti\goat 488 staining on B cells with beads. The graphs below the plots show the phagocytic index of WT and = 3). Phagocytic index for WT B cells incubated for 1 h with 1, 3, and Dimebon 2HCl 10 m beads coated with anti\IgM. Data symbolize means SEM (= 3). Confocal section and orthogonal images of follicular WT and = 3). Proliferation profiles of OT2 T cells after 3 days of culture with WT (black) or = 3). Data information: *< 0.05; **< 0.005; ***< 0.0005 (unpaired Student's through an actin\ and RhoG\dependent mechanism Follicular B cells phagocytose particulate antigens through a RhoG\dependent mechanism. Circulation cytometry plots of purified FO B cells incubated for 1 h at 0C or 37C with 1 m fluorescent beads coated with a goat anti\mouse anti\IgM antibody and stained afterwards extracellularly on ice with an anti\goat IgG 488. Gate shows the B cells with internalized beads (unfavorable for the anti\goat IgG staining). Circulation.